Project Summary Humans, like all mammals, possess limited natural ability to efficiently replace lost myocardium with new contractiletissue.Thisdeficiencycontributestoheartfailure,theleadingcauseofmorbidityandmortalityinthe United States. By contrast with mammals, teleost fish spontaneously and robustly regenerate new cardiac tissues after heart injury, representing a useful model for heart repair and regeneration. We are interested to understand how regenerative responses to injury have been optimized in non-mammalian vertebrates like zebrafish,todiscovernewtargetsthatunderlietheregenerativedeficienciesinmammals.Wehaveinvestigated theregenerativebiologyoftwomajoradultcardiactissues:themyocardiumandtheepicardium.Wefoundthat adultzebrafishcanfullyregeneratelostmyocardiumandreversethesignsofheartfailurewithinseveralweeks. Wealsofoundthattheepicardiumisrequiredformyocardialregenerationandvigorouslyregeneratesafter90% lossofitself,withabase-apexdirectionality,thatiscompletedin2weeks.Currently,wearedefiningtheroleof perivascularcellsandthemechanismofcoronaryrevascularizationduringheartregeneration.Themajorhurdle tostudythecoronaryvasculatureisthelackofgenetictoolstospecificallymarkandmanipulateitsmajorcellular components: perivascular cells and coronary endothelial cells. In preliminary studies, we employed deep sequencing,insituhybridizationandBACtransgenictechnologyinsearchfornovelgeneticmarkersspecificfor theseelusivecelltypesandcandidategenesup-regulatedafterheartinjury.Wehaveidentifiedanewtransgenic straintospecificallymarkperivascularcells,andanoveltransgenicstrainspecificallylabelscoronaryvessels. Withbunchofnewtoolsandnewly-modifiedexvivosystemforthisproposal,wefoundthatperivascularcells may provide cellular clues for growing vessels during initial formation of the coronary vascularization and that they rapidly accumulate in the sites of cardiac wounds prior to revascularization and regeneration. In this proposal, we will address central questions about the requirements of perivascular cells for myocardial regenerationandcoronaryrevascularization.Ouroverallhypothesisisthatavigorousperivascularcellresponse iscriticalformyocardialregenerationandcoronaryrevascularization.Totestthishypothesis,wewill:1)define the requirements of perivascular cells during heart regeneration with new methods of depleting perivascular cells,andcharacterizetheregenerativebiologyofperivascularcellswithlineage-tracingexperiments;?2)define the cardiac regenerative responses to the coronary damage with new genetic injury methods, and perform chemicalscreenstoidentifyregulatorsofcoronaryrevascularization;?and3)definetheroleofperivascularcells for guiding vessel growth in coronary revascularization during heart regeneration. Our work will generate paradigm-shifting discoveries in perivascular cell and coronary vascular biology. It will reveal the impact of perivascular cells on heart regeneration and key underlying regulators. These findings will inform approaches forcomprehendingandenhancingthelimitedregenerationdisplayedbyhumansaftermyocardialinfarction(MI).